Compositions And Methods Of Altering The Electric Impedance To An Alternating Electric Field

ABSTRACT

Disclosed are methods of altering the electric impedance to an alternating electric field in a target site of a subject, comprising introducing a nanoparticle to a target site in the subject; and applying an alternating electric field to the target site of the subject, wherein the electric impedance in the target site of the subject to the alternating current is altered. Disclosed are methods for improving transport of a nanoparticle across a cell membrane of a cell, the method comprising applying an alternating electric field to the cell for a period of time, wherein application of the alternating electric field increases permeability of the cell membrane; and introducing the nanoparticle to the cell, wherein the increased permeability of the cell membrane enables the nanoparticle to cross the cell membrane. Disclosed are methods of imaging a cancer cell, the method comprising applying a first alternating electric field at a first frequency to the cancer cell for a first period of time, wherein application of the first alternating electric field at the first frequency to the cancer cell for the first period of time increases permeability of cell membranes of the cancer cell; introducing a nanoparticle to the cancer cell, wherein the increased permeability of the cell membranes enables the nanoparticle to cross the cancer cell membrane; and imaging the cancer cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/946,798 filed Dec. 11, 2019 and is hereby incorporated herein byreference in its entirety.

BACKGROUND

Tumor Treating Fields, or TTFields, are typically low intensity (e.g.,1-3 V/cm) alternating electric fields within the intermediate frequencyrange (100-300 kHz). TTFs through can deliver alternating electricfields through non-invasive transducer arrays across the anatomicalregion of a tumor. TTFs have been established as an anti-mitotic cancertreatment modality because they interfere with proper micro-tubuleassembly during metaphase and eventually destroy the cells duringtelophase, cytokinesis, or subsequent interphase. TTFields have beenshown to not affect the viability of non-dividing normal cells, nerves,and muscles because of their low intensity. TTField therapy is anapproved mono-treatment for recurrent glioblastoma, and an approvedcombination therapy with chemotherapy for newly diagnosed glioblastomaand unresectable malignant pleural mesothelioma patients. These electricfields are induced non-invasively by transducer arrays (i.e., arrays ofelectrodes) placed directly on the patient's scalp. TTFields also appearto be beneficial for treating tumors in other parts of the body.

BRIEF SUMMARY

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprisingintroducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprising:introducing a non-conductive nanoparticle to a site adjacent to thetarget site in the subject; introducing a conductive nanoparticle to atarget site in the subject; applying an alternating electric field tothe target site and the site adjacent to the target site of the subject,wherein the efficacy of the alternating electric field in the targetsite of the subject is increased.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprising:introducing a non-conductive nanoparticle to a site adjacent to thetarget site in the subject; applying an alternating electric field tothe site adjacent to the target site or to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased.

Disclosed are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprisingintroducing a nanoparticle to a target site in the subject; and applyingan alternating electric field to the target site of the subject, whereinthe electric impedance in the target site of the subject to thealternating current is altered.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a nanoparticle to a target site in the subject; applying analternating electric field to the target site of the subject, whereinthe efficacy of the alternating electric field in the target site of thesubject is increased.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a non-conductive nanoparticle to a non-target site adjacentto the target site in the subject; applying an alternating electricfield to the target site of the subject, wherein the efficacy of thealternating electric field in the target site of the subject isincreased.

Disclosed are methods for improving transport of a nanoparticle across acell membrane of a cell, the method comprising applying an alternatingelectric field to the cell for a period of time, wherein application ofthe alternating electric field increases permeability of the cellmembrane; and introducing the nanoparticle to the cell, wherein theincreased permeability of the cell membrane enables the nanoparticle tocross the cell membrane.

Disclosed are methods for reducing the viability of cancer cells, themethod comprising: applying a first alternating electric field at afirst frequency to the cancer cells for a first period of time, whereinapplication of the first alternating electric field at the firstfrequency to the cancer cells for the first period of time increasespermeability of cell membranes of the cancer cells; introducing ananoparticle to the cancer cells, wherein the increased permeability ofthe cell membranes enables the nanoparticle to cross the cell membranes;and applying a second alternating electric field at a second frequencyto the cancer cells for a second period of time, wherein the secondfrequency is different from the first frequency, and wherein the secondalternating electric field at the second frequency reduces viability ofthe cancer cells.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show exemplary effects ofenhanced tumor conductivity on TTFields intensity: (FIG. 1A) Axialslices of an MRI of a patient with GBM, with his gross tumor volumeregion marked (FIG. 1B) Simulation results of a computerized head modelresulted from assigning the conductivity of the enhancing tumor tissue avalue 0.24 S/m (FIG. 1C) Simulation results of a computerized head modelresulted from assigning the conductivity of the enhancing tumor tissue avalue 0.3 S/m (FIG. 1D) Relative difference of simulation results of 0.3S/m vs. 0.24 S/m.

FIG. 2 shows a histogram of average LMiPD in the gross tumor volume of45 head models of GBM patients treated with TTFields.

FIG. 3A, FIG. 3B, and FIG. 3C show physicochemical characterization ofBTNPs. (FIG. 3A) FE-SEM and (FIG. 3B) TEM images of BTNPs. (FIG. 3C)Sizes and zeta-potential values of FBS coated BTNPs.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F showcytocompatibility of BTNPs in breast cancer cells. (FIG. 4A and FIG. 4B)Cell proliferation, (FIG. 4C and FIG. 4D) representative images fromclonogenic assays, and (FIG. 4E and FIG. 4F) colony counting in MCF-7and BT-549 cells upon BTNP treatment. Data represent mean±standarddeviation of three independent experiments; **P<0.01, and *P<0.05. N.S.not significant.

FIG. 5A, FIG. 5B, and FIG. 5C show BTNPs enhanced the antitumor activityof TTFields in low TTField-sensitive MCF-7 cells. (FIG. 5A) Cellproliferation following TTFields in MCF-7, MDA-MB-231, and BT-549 cells,(FIG. 5B) Relative number of cells with TTFields or TTFields and BTNPstreatment to MCF-7 cells, and (FIG. 5C) quantification of colonies. Datarepresent mean±standard deviation of three independent experiments;**P<0.01, and *P<0.05.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E show cytoplasmicaccumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields.(FIG. 6A and FIG. 6B) Flow cytometry histogram and (FIG. 6C and FIG. 6D)representative images showing cytosolic localization of BTNPs in MCF-7and BT-549 cells treated with TTFields or TTFields and BTNP. (FIG. 6E)TEM images confirming the cytosolic localization of BTNPs inTTField-treated MCF-7 cells. Data is representative of three independentexperiments.

FIG. 7A and FIG. 7B show changes in gene copy number in MCF-7 cells onTTFields and BTNP combinatorial treatment. (FIG. 7A) Heatmap with globalsignificance scores and global significance statistics and (FIG. 7B)directed global significance scores for cells treated with TTFields orTTFields and BTNPs.

FIG. 8A and FIG. 8B show modulation of cell cycle-apoptosis pathways byBTNPs combined with TTFields. (FIG. 8A) Gene signatures related to cellcycle pathways grouped in a heatmap and (FIG. 8B) Western blot of MCF-7cells treated with TTFields or TTFields and BTNP. Data is representativeof three independent experiments.

FIG. 9 shows a schematic representation of the proposed mechanism ofcancer cell sensitization induced by BTNPs in presence of TTFields.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Examples included therein and to the Figures andtheir previous and following description.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. Thus, if a class of molecules A, B, and C are disclosed as wellas a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited, each is individually and collectively contemplated. Thus, isthis example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D,C-E, and C-F are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. Thus, for example, thesub-group of A-E, B-F, and C-E are specifically contemplated and shouldbe considered disclosed from disclosure of A, B, and C; D, E, and F; andthe example combination A-D. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods, and that each suchcombination is specifically contemplated and should be considereddisclosed.

A. Definitions

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ananoparticle” includes a single or a plurality of such nanoparticles,reference to “the nanoparticle” is a reference to one or morenanoparticles and equivalents thereof known to those skilled in the art,and so forth.

As used herein, a “target site” is a specific site or location within orpresent on a subject or patient. For example, a “target site” can referto, but is not limited to a cell, population of cells, organ, tissue,tumor, or cancer cell. In some aspects, organs include, but are notlimited to, lung, brain, pancreas, abdominal organs (e.g. stomach,intestine), ovary, breast, uterus, prostate, bladder, liver, colon, orkidney. In some aspects, a cell or population of cells include, but arenot limited to, lung cells, brain cells, pancreatic cells, abdominalcells, ovarian cells, liver cells, colon cells, or kidney cells. In someaspects, a “target site” can be a tumor target site.

A “tumor target site” is a site or location within or present on asubject or patient that comprises or is adjacent to one or more cancercells, previously comprised one or more tumor cells, or is suspected ofcomprising one or more tumor cells. For example, a tumor target site canrefer to a site or location within or present on a subject or patientthat is prone to metastases. Additionally, a target site or tumor targetsite can refer to a site or location of a resection of a primary tumorwithin or present on a subject or patient. Additionally, a target siteor tumor target site can refer to a site or location adjacent to aresection of a primary tumor within or present on a subject or patient.

As used herein, an “alternating electric field” or “alternating electricfields” refers to a very-low-intensity, directional,intermediate-frequency alternating electrical fields delivered to asubject, a sample obtained from a subject or to a specific locationwithin a subject or patient (e.g. a target site or a tumor target site).In some aspects, the alternating electrical field can be in a singledirection or multiple directional.

An example of an alternating electric field includes, but is not limitedto, a tumor-treating field. In some aspects, TTFields can be deliveredthrough two pairs of transducer arrays that generate perpendicularfields within the treated tumor. For example, for the Optune™ system (aTTField delivery system) one pair of electrodes is located to the leftand right (LR) of the tumor, and the other pair of electrodes is locatedanterior and posterior (AP) to the tumor. Cycling the field betweenthese two directions (i.e., LR and AP) ensures that a maximal range ofcell orientations is targeted.

As described herein, TTFields have been established as an anti-mitoticcancer treatment modality because they interfere with propermicro-tubule assembly during metaphase and eventually destroy the cellsduring telophase, cytokinesis, or subsequent interphase. TTFields targetsolid tumors and is described in U.S. Pat. No. 7,565,205, which isincorporated herein by reference in its entirety for its teaching ofTTFields.

In-vivo and in-vitro studies show that the efficacy of TTFields therapyincreases as the intensity of the electrical field increases. Therefore,optimizing array placement on the patient's scalp to increase theintensity in the diseased region of the brain is standard practice forthe Optune system. Array placement optimization may be performed by“rule of thumb” (e.g., placing the arrays on the scalp as close to thetumor as possible), measurements describing the geometry of thepatient's head, tumor dimensions, and/or tumor location. Measurementsused as input may be derived from imaging data. Imaging data is intendedto include any type of visual data, such as for example, single-photonemission computed tomography (SPECT) image data, x-ray computedtomography (x-ray CT) data, magnetic resonance imaging (MRI) data,positron emission tomography (PET) data, data that can be captured by anoptical instrument (e.g., a photographic camera, a charge-coupled device(CCD) camera, an infrared camera, etc.), and the like. In certainimplementations, image data may include 3D data obtained from orgenerated by a 3D scanner (e.g., point cloud data). Optimization canrely on an understanding of how the electrical field distributes withinthe head as a function of the positions of the array and, in someaspects, take account for variations in the electrical propertydistributions within the heads of different patients. The term “subject”refers to the target of administration, e.g. an animal. Thus, thesubject of the disclosed methods can be a vertebrate, such as a mammal.For example, the subject can be a human. The term does not denote aparticular age or sex. Subject can be used interchangeably with“individual” or “patient.” For example, the target of administration canmean the recipient of the alternating electrical field.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinence ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

B. Nanoparticles

Disclosed herein are methods involving nanoparticles. Any of thenanoparticles described herein can be used for one or more of thedisclosed methods.

In some aspects, the nanoparticle can comprise a conducting orsemi-conducting material. For example, the nanoparticle can comprise orconsist of carbon gold, ferrous iron, selenium, silver, copper,platinum, iron oxide, graphene, iron dextran, superparamagnetic ironoxide, boron-doped detonation nanodiamonds, or a combination thereof. Insome aspects, the nanoparticle can comprise an alloy selected fromAu/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Fe, Au/Cu or Au/Fe/Cu.

In some aspects, the nanoparticle can be a conductive nanoparticle. Aconductive nanoparticle can increase conductivity and lower impedance ina target site or tumor target site. Thus, in some aspects of thedisclosed methods, the impedance in a target site or tumor target siteis lowered and/or the conductivity in a target site or tumor target siteis increased.

In some aspects, the nanoparticle is a non-conductive nanoparticle. Insome aspects, the non-conductive nanoparticle is a ferroelectricnanoparticle. Ferroelectric nanoparticles have emerged as promisingtools for enhancing electric stimulation of cells and tissues. Severalnanotransducers have been revealed to mediate photodynamic andmagnetothermal conversions, and to locally deliver anticancer stimuli totumor burden in the field of nanooncology. Cell and tissue penetrationof these nanotransducers could be controlled by remote electricalstimulation. Among ferroelectric nanoparticles, barium titanatenanoparticles (BTNPs) have high dielectric constants and suitablepiezoelectric characteristics with high biocompatibility. Suchnon-conductive nanoparticles can be used in the methods disclosed hereinto be taken up by a cell via TTField stimulation and to promote theantitumor action of TTFields by enhancing cell cycle-related apoptosisin cancer cells. In some aspects, the non-conductive nanoparticle is nota ferroelectric nanoparticle. A non-conductive nanoparticle can decreaseconductivity and increase impedance in a target site or tumor targetsite. Thus, in some aspects of the disclosed methods, the impedance in atarget site or tumor target site is increased and/or the conductivity ina target site or tumor target site is decreased.

In some aspects, a population of nanoparticles can be used in themethods disclosed herein. In some aspects, the population ofnanoparticles can include conductive and non-conductive nanoparticles.

Nanoparticles (NPs) internalization into cells is known to be dependenton particle size and its zeta potential. NPs under 200 nm can beengulfed by cancer cells through clathrin-dependent pathway ormacro-pinocytosis pathway. In some aspects, the size of the nanoparticlecan be between 0.5 nm and 100 nm. In some aspects, the size of thenanoparticle can be between 0.5 nm & 2.5 nm. In some aspects, the sizeof the nanoparticle can be between 100 nm and 200 nm. In some aspects,the size of the nanoparticle can be greater than 100 nm. In someaspects, the disclosed methods allow for the use of nanoparticles (e.g.metal/magnetic), in a size range of 100 nm-200 nm (preferentially up to150 nm to avoid accumulation in the liver and spleen), to target cancercells in vivo.

In some aspects, the nanoparticle has a three-dimensional shape. Forexample, the nanoparticle can be a nanocube, nanotube, NanoBipyramid,NanoPlate, NanoCluster, Nanochaine, NanoStar, NanoShuttle, NanoHollow,dendrimer, nanorod, nanoshell, nanocage, nanosphere, nanofiber, ornanowire, or a combination thereof.

In some aspects, the nanoparticle can be mesoporous or nonporous.

In some aspects, the nanoparticle can be coated with a polysaccharide,poly amino acid, or synthetic polymer. Suitable coating for thenanoparticle can be chosen to decrease the toxicity of the nanoparticleand can provide the nanoparticle with the capacity for selectiveinteraction with different types of cells and biological molecules.Suitable coating for the nanoparticle can be chosen to improve thenanoparticle biocompatibility and solubility in water and biologicalfluids by decreasing their aggregation capacity or increasing theirstability, Suitable coating for the nanoparticle can be chosen toinfluence the nanoparticle pharmacokinetics, changing the patterns ofthe nanoparticle and/or distribution and accumulation in the body.

In some aspects, the nanoparticles can be incorporated into a scaffoldprior to introducing the nanoparticles to the subject. In some aspects,the nanoparticles can be loaded onto or within a scaffold prior to orafter introducing the scaffold to a subject. For example, a scaffoldcould be surgically provided to a subject and subsequently one or moreof the nanoparticles described herein could be administered to thesubject under conditions that allow for the nanoparticles to incorporateinto the scaffold. Alternatively, nanoparticles could be incorporatedinto a scaffold outside of a subject and then the nanoparticle loadedscaffold could be surgically provided to a subject.

Examples of scaffolds include, but are not limited to scaffoldscomprising natural polymers such as hyaluronic acid, fibrin, chitosan,and collagen. Examples of scaffolds include, but are not limited toscaffolds comprising synthetic polymers such as Polyethylene Glycol(PEG), polypropylene fumarate (PPF), polyanhydride, polycaprolactone(PCL), polyphosphazene, polyether ether ketone (PEEK), polylactic acid(PLA), and poly (glycolic acid) (PGA).

In some aspects, the nanoparticle is conjugated to one or more ligands.In some aspects, the one or more ligands can be conjugated to thenanoparticle via a linker. In some aspects, a linker comprises a thiolgroup, a C2 to C12 alkyl group, a C2 to C12 glycol group or a peptide.In some aspects, the linker comprises a thiol group represented by thegeneral formula HO—(CH)n,—S—S—(CH2)m-OH wherein n and m areindependently between 1 and 5. In some aspects, the one or more ligandsare a small molecule, nucleic acid, carbohydrate, lipid, peptide,antibody, antibody fragment, or a therapeutic agent. For example the oneor more ligands can be, but are not limited to, an anticancer drug, acytotoxic drug, pain-management drug, pseudomonas exotoxin A, anon-radioactive isotope (e.g. boron-10 for boron neutron capturetherapy), or a photosensitizer (e.g. photofrin, foscan, 5-aminolevulinicacid, Mono-L-aspartyl chlorin e6, pthalocyanines,Meta-tetra(hydroxyphenyl)porphyrins, texaphyrins, Tin ethyletipurpurin).

In some aspects, the nanoparticle can be a labeled nanoparticle. In someaspects, labeled nanoparticles can be magnetic nanoparticles,nanoparticles decorated with Gd3+, nanoparticles decorated radioisotopes(e.g. technetium-99m, iodine-123, iodine-131, fluorine-18 carbon-11,nitrogen-13, oxygen-15, gallium-68, zirconium-89, and rubidium-82),nanoparticles decorated with a fluorescent label (e.g. Quantum dots),nanoparticles decorated with photosensitizer (e.g. photofrin, foscan,5-aminolevulinic acid, Mono-L-aspartyl chlorin e6, pthalocyanines,Meta-tetra(hydroxyphenyl)porphyrins, texaphyrins, Tin ethyletipurpurin), nanoparticles decorated with dye. In some aspects, thenanoparticle can be coated with a labeled antibody and therefore thenanoparticle is indirectly labeled. In some aspects, if there are sizeconstraints, the nanoparticles, if decorated or conjugated to a largemoiety, can be of a smaller size to accommodate a larger moiety.

Other examples of nanoparticles include, but are not limited to, silicananoparticles, hydrophilic polymers (e.g. polyacrylamide (PAA),polyurethanes, poly(hydroxyethyl methacrylamide) (pHEMA), certainpoly(ethylene glycols), and hydrophobic polymers (e.g. polystyrenenanoparticles).

In some aspects, the nanoparticle can be introduced into a target site.In some aspects, the nanoparticle can be introduced into a tumor targetsite. In some aspects, the nanoparticle can be introduced into a tumoror cancer cell. In some aspects, the nanoparticle can be introduced intoa location in a subject suspected of comprising one or more tumor cells.In some aspects, the nanoparticle can be introduced into to a site orlocation within or present on a subject or patient that is prone tometastases. In some aspects, the nanoparticle can be introduced into toa site or location of a resection of a primary tumor within or presenton a subject or patient. In some aspects, the nanoparticle can beintroduced into the tumor or cancer cell via injection post primarytumor resection. In some aspects, the nanoparticle can be introducedinto a site adjacent to a target site. In some aspects, the nanoparticlecan be introduced into a site adjacent to a location in a subjectsuspected of comprising one or more tumor cells.

In some aspects, the nanoparticle can be introduced into a tumor targetsite, wherein the tumor target site adjacent to a tumor target site. Insome aspects, the nanoparticle can be introduced into a tumor targetsite, wherein the tumor target site is adjacent to a tumor or cancercell. In some aspects, the nanoparticle can be introduced into a tumortarget site, wherein the tumor target site adjacent to a location in asubject suspected of comprising one or more tumor cells. In someaspects, the nanoparticle can be introduced into a tumor target site,wherein the tumor target site adjacent to a site or location within orpresent on a subject or patient that is prone to metastases. In someaspects, the nanoparticle can be introduced into a tumor target site,wherein the tumor target site adjacent to a site or location of aresection of a primary tumor within or present on a subject or patient.In some aspects, the nanoparticle can be introduced into a tumor targetsite, wherein the tumor target site adjacent to the tumor or cancer cellvia injection post primary tumor resection. In some aspects, thenanoparticle can be introduced into a tumor target site, wherein thetumor target site adjacent to the tumor or cancer cell via intratumorinjection (e.g. computed tomography-guided, during surgery or biopsy).

In some aspects, the nanoparticle can be introduced intratumorally,intracranially, intraventricularly, intrathecally, epidurally,intradurally, intravascularly, intravenously (targeted or non-targeted),intraarterially, intramuscularly, subcutaneously, intraperitoneally,orally, intranasally, via intratumor injection (e.g. computedtomography-guided, during surgery or biopsy) or via inhalation. In someaspects, nanoparticles can be targeted to the tumor or tumor target siteusing tumor-targeting moieties. Tumor-targeting moieties can be, but arenot limited to, folate, transferrin, aptamers, antibodies, nucleic acidsand peptides. Thus, in some aspects, the nanoparticle can be introducedto the subject in a targeted or non-targeted manner.

In some aspects, the nanoparticle can be introduced at a concentrationbased on tumor volume, method of delivery, limitations of the deviceadministering the alternating electric field, patient weight, patientage, size of tumor, the type of tumor or cancer, location of the tumoror cancer cells, age of the patient, or any other physical or genotypicattribute of the patient, cancer cell or tumor. In some aspects, thesize of the nanoparticles can be used to determine the concentration ofthe nanoparticles to be introduced. In some aspects, the nanoparticlecan be introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300,300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900,or 900 to 1000 ng per mm³ tumor. In some aspects, the nanoparticle canbe introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5,5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900to 1000 μg.

In some aspects, the nanoparticle can be introduced to the subject once,twice, or three or more times.

1. Pharmaceutical Compositions

Disclosed herein are pharmaceutical compositions comprising one or moreof the nanoparticles described herein. In some aspects, thenanoparticles described herein can be provided in a pharmaceuticalcomposition. For example, the nanoparticles described herein can beformulated with a pharmaceutically acceptable carrier.

In some aspects, a pharmaceutical composition can comprise achemotherapeutic agent. In some aspects, a pharmaceutical compositioncan comprise a chemotherapeutic agent and one or more of thenanoparticles described herein. For example, disclosed herein arepharmaceutical compositions comprising one or more of the nanoparticlesdescribed herein and an anticancer drug, a cytotoxic drug,pain-management drug, pseudomonas exotoxin A, a non-radioactive isotope(e.g. boron-10 for boron neutron capture therapy), and/or aphotosensitizer (e.g. photofrin, foscan, 5-aminolevulinic acid,Mono-L-aspartyl chlorin e6, pthalocyanines,Meta-tetra(hydroxyphenyl)porphyrins, texaphyrins, Tin ethyletipurpurin).

Disclosed herein are compositions comprising one or more of thenanoparticles described herein that that further comprise a carrier suchas a pharmaceutically acceptable carrier. For example, disclosed arepharmaceutical compositions, comprising the nanoparticles disclosedherein, and a pharmaceutically acceptable carrier.

For example, the nanoparticles described herein can comprise apharmaceutically acceptable carrier. By “pharmaceutically acceptable” ismeant a material or carrier that would be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart. Examples of carriers include dimyristoylphosphatidyl (DMPC),phosphate buffered saline or a multivesicular liposome. For example,PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in thisinvention. Other suitable pharmaceutically acceptable carriers and theirformulations are described in Remington: The Science and Practice ofPharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton,Pa. 1995. Typically, an appropriate amount ofpharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Other examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutioncan be from about 5 to about 8, or from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semi-permeablematrices of solid hydrophobic polymers containing the composition, whichmatrices are in the form of shaped articles, e.g., films, stents (whichare implanted in vessels during an angioplasty procedure), liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of nanoparticlebeing administered. These most typically would be standard carriers foradministration of drugs to humans, including solutions such as sterilewater, saline, and buffered solutions at physiological pH.

Pharmaceutical compositions can also include carriers, thickeners,diluents, buffers, preservatives and the like, as long as the intendedactivity of the polypeptide, peptide, nucleic acid, vector of theinvention is not compromised. Pharmaceutical compositions may alsoinclude one or more active ingredients (in addition to the compositionof the invention) such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutical composition may beadministered in a number of ways depending on whether local or systemictreatment is desired, and on the area to be treated.

Preparations of parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for optical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids, or binders may be desirable. Some of the compositionsmay potentially be administered as a pharmaceutically acceptable acid-or base-addition salt, formed by reaction with inorganic acids such ashydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acidssuch as formic acid, acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleicacid, and fumaric acid, or by reaction with an inorganic base such assodium hydroxide, ammonium hydroxide, potassium hydroxide, and organicbases such as mon-, di-, trialkyl and aryl amines and substitutedethanolamines.

In the methods described herein, delivery (or administration orintroduction) of the nanoparticles or pharmaceutical compositionsdisclosed herein to subjects can be via a variety of mechanisms.

C. Altering Impedance in a Target Site

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered, wherein the current density and/or powerloss density in the target site of the subject to the alternatingcurrent is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the conductivity is decreasedin the site adjacent to the target site.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the impedance in the siteadjacent to the target site is increased.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the conductivity is increasedin the target site.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the impedance in the targetsite is decreased.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the non-conductivenanoparticle is not a ferroelectric nanoparticle.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered and wherein the method further comprisesintroducing a nanoparticle to a target site in the subject and applyingan alternating electric field to the target site of the subject. In someaspects, the impedance in the target site is lowered and/or theconductivity in the target site is increased.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered, wherein the nanoparticle is anon-conductive nanoparticle. In some aspects, the impedance in thetarget site is increased and/or conductivity in the target site isdecreased.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered, wherein the alternating electric fieldis a tumor-treating field.

Disclosed are methods of altering the electric impedance to analternating electric field in a site adjacent to a target site of asubject, comprising: introducing a non-conductive nanoparticle to a siteadjacent to the target site in the subject; and applying an alternatingelectric field to the site adjacent to the target site of the subject,wherein the electric impedance in the site of the subject to thealternating current is altered, wherein the target site is a tumortarget site. In some aspects, the altered electric impedance in thetarget site of the subject to the alternating current results in anincreased mitotic effect of the alternating electric field in the targetsite.

Disclosed herein are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprising:introducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered.

Disclosed herein are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprising:introducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered, wherein the currentdensity and/or power loss density in the target site of the subject tothe alternating current is altered, wherein the impedance in the targetsite is lowered and/or wherein the conductivity in the target site isincreased.

Disclosed herein are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprising:introducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered, wherein the currentdensity and/or power loss density in the target site of the subject tothe alternating current is altered, wherein the method further comprisesintroducing a non-conductive nanoparticle to a site adjacent to thetarget site in the subject; and applying an alternating electric fieldto the site adjacent to the target site of the subject. In some aspects,the current density and/or power loss density in the target site of thesubject to the alternating current is altered. In some aspects, theconductivity is decreased in the site adjacent to the target site. Insome aspects, the impedance in the site adjacent to the target site isincreased. In some aspects, the conductivity is increased in the targetsite. In some aspects, the impedance in the target site is decreased.

Disclosed herein are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprising:introducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered, wherein the currentdensity and/or power loss density in the target site of the subject tothe alternating current is altered, wherein the non-conductivenanoparticle is not a ferroelectric nanoparticle. In some aspects, theimpedance in the target site is increased and/or the conductivity in thetarget site is decreased.

Disclosed herein are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprising:introducing a conductive nanoparticle to a target site in the subject;and applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered, wherein the currentdensity and/or power loss density in the target site of the subject tothe alternating current is altered, wherein the alternating electricfield is a tumor-treating field. In some aspects, the nanoparticles arenanoparticles that increase tissue permittivity. In some aspects, thetarget site is a tumor target site. In some aspects, the alteredelectric impedance in the tumor target site of the subject to thealternating current results in an increased mitotic effect of thealternating electric field in the tumor target site.

Disclosed are methods of altering the electric impedance to analternating electric field in a target site of a subject, comprisingintroducing a nanoparticle to a target site in the subject; and applyingan alternating electric field to the target site of the subject, whereinthe electric impedance in the target site of the subject to thealternating current is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a tumor target site of a subject,comprising introducing a nanoparticle to a tumor target site in thesubject; and applying an alternating electric field to the tumor targetsite of the subject, wherein the electric impedance in the tumor targetsite of the subject to the alternating current is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a tumor target site of a subject,comprising introducing a nanoparticle to a tumor target site in thesubject; and applying an alternating electric field to the tumor targetsite of the subject, wherein the electric impedance in the tumor targetsite of the subject to the alternating current is altered, wherein thetumor target site is adjacent to one or more cancer cells, previouslycomprised one or more tumor cells, or is suspected of comprising one ormore tumor cells. Disclosed are methods of altering the electricimpedance to an alternating electric field in a target site of asubject, comprising introducing a nanoparticle to a target site in thesubject; and applying an alternating electric field to the target siteof the subject, wherein the electric impedance in the target site of thesubject to the alternating current is altered, wherein the currentdensity and/or power loss density in the target site of the subject tothe alternating current is altered. Disclosed are methods of alteringthe electric impedance to an alternating electric field in a tumortarget site of a subject, comprising introducing a nanoparticle to atumor target site in the subject; and applying an alternating electricfield to the tumor target site of the subject, wherein the electricimpedance in the tumor target site of the subject to the alternatingcurrent is altered, wherein the current density and/or power lossdensity in the tumor target site of the subject to the alternatingcurrent is altered.

Disclosed are methods of altering the electric impedance to analternating electric field in a tumor target site of a subject,comprising introducing a nanoparticle to a tumor target site in thesubject; and applying an alternating electric field to the tumor targetsite of the subject, wherein the electric impedance in the tumor targetsite of the subject to the alternating current is altered, wherein thecurrent density and/or power loss density in the tumor target site ofthe subject to the alternating current is altered, wherein the tumortarget site is adjacent to one or more cancer cells, previouslycomprised one or more tumor cells, or is suspected of comprising one ormore tumor cells.

In some aspects, the current density in the target site or tumor targetsite is increased. In some aspects, the current density is in the targetsite or tumor target site decreased. In some aspects, power loss densityin the target site or tumor target site is increased. In some aspects,power loss density in the target site or tumor target site is decreased.

In some aspects, the nanoparticle is a conductive nanoparticle. In someaspects, a conductive nanoparticle can increase conductivity and lowerimpedance in the target site or tumor target site. Thus, in some aspectsof the disclosed methods, the impedance in the target site or tumortarget site is lowered and/or the conductivity in the target site ortumor target site is increased.

In some aspects, the nanoparticle is a non-conductive nanoparticle. Insome aspects, the non-conductive nanoparticle is not a ferroelectricnanoparticle. In some aspects, a non-conductive nanoparticle candecrease conductivity and increase impedance in the target site or tumortarget site. Thus, in some aspects of the disclosed methods, theimpedance in the target site or tumor target site is increased and/orthe conductivity in the target site or tumor target site is decreased.

In some aspects, a population of nanoparticles can be used in themethods disclosed herein. In some aspects, the population ofnanoparticles can include conductive and non-conductive nanoparticles.

In some aspects, the alternating electric field used in the methodsdisclosed herein is a tumor-treating field. In some aspects, thealternating electric field (e.g. tumor-treating field) can varydependent on the type of cancer or tumor being treated. In some aspects,the cancer cells are glioblastoma cells, uterine sarcoma cells, breastadenocarcinoma cells, pancreatic cancer cells, non-small cell lungcancer, hepatocellular, gastric cancer cells, brain cancer cells kidneycancer cells, neuroblastoma cells, colon cancer cells, bladder cancercells, prostate cancer cells, or thymus cancer cells. In some aspects,the frequency of the alternating electric fields can be 200 kHz. Thefrequency of the alternating electric fields can also be, but is notlimited to, about 200 kHz, between 50 and 500 kHz, between 100 and 500kHz, between 25 kHz and 1 MHz, between 50 and 190 kHz, between 25 and190 kHz, or between 210 and 400 kHz.

In some aspects, the field strength of the alternating electric fieldscan be between 1 and 4 V/cm RMS. In some aspects, different fieldstrengths can be used (e.g., between 0.1 and 10 V/cm).

In some aspects, the alternating electric fields can be applied for avariety of different intervals ranging from 0.5 hours to 72 hours. Insome aspects, a different duration can be used (e.g., between 0.5 hoursand 14 days). In some aspects, application of the alternating electricfields can be repeated periodically. For example, the alternatingelectric fields can be applied every day for a two hour duration.

In some aspects, the nanoparticles are nanoparticles that increasetissue or cell permittivity.

In some aspects, the altered electric impedance in the target site ortumor target site of the subject to the alternating current results inan increased anti mitotic effect of the alternating electric field inthe target site. For example, the increased anti mitotic effect canrefer to interference with proper micro-tubule assembly during metaphasewhich can eventually destroy the cells (e.g. cancer cells) present in orat the target site during telophase, cytokinesis, or subsequentinterphase.

Disclosed are methods of altering the electric impedance in a targetsite or tumor target site with one frequency that allows nanoparticlesto enter into cells in the target site or tumor target site and thenapplying a second frequency to the target site or tumor target sitewherein the electric impedance to the second frequency in the targetsite or tumor target site is altered. Disclosed are methods of alteringthe electric impedance in a target site or tumor target site with onefrequency (a first frequency) that allows nanoparticles to enter intocells in the target site or tumor target site and then applying a secondfrequency to the target site or tumor target site wherein the electricimpedance to the second frequency in the target site or tumor targetsite is altered, further comprising applying multiple first and secondfrequencies. For example, disclosed are methods of altering the electricimpedance to an alternating electric field in a target site or tumortarget site of a subject, comprising applying a first alternatingelectric field at a first frequency to the target site or tumor targetsite for a first period of time, wherein application of the firstalternating electric field at the first frequency to the target site ortumor target site for the first period of time increases permeability ofcell membranes of the cells present in the target site or tumor targetsite; introducing a nanoparticle to the target site or tumor targetsite, wherein the increased permeability of the cell membranes enablesthe nanoparticle to cross the cell membranes; and applying a secondalternating electric field at a second frequency to the target site ortumor target site for a second period of time, wherein the secondfrequency is different from the first frequency, and wherein theimpedance in the target site or tumor target site of the subject of thesecond alternating electric field at the second frequency is altered. Insome aspects, the current density and/or power loss density in thetarget site or tumor target site of the subject to the alternatingcurrent is altered. In some aspects, the cells are cancer cells. In someaspects, the cancer cells are glioblastoma cells, uterine sarcoma cells,breast adenocarcinoma cells, pancreatic cancer cells, non-small celllung cancer, hepatocellular, gastric cancer cells, or brain cancercells. In some aspects, the cancer cells comprise glioblastoma cells,the first frequency is between 250 kHz and 350 kHz, and the secondfrequency is between 150 kHz and 250 kHz. In some aspects, the cancercells comprise uterine sarcoma cells, the first frequency is between 125kHz and 175 kHz, and the second frequency is between 75 kHz and 125 kHz.In some aspects, the cancer cells comprise breast adenocarcinoma cells,the first frequency is between 75 kHz and 175 kHz, and the secondfrequency is between 100 kHz and 300 kHz. In some aspects, the step ofintroducing the nanoparticle begins at a given time, and wherein thestep of applying the first alternating electric field ends at least 12hours after the given time. In some aspects, the step of applying thefirst alternating electric field begins at least one hour before thegiven time. In some aspects, the second period of time comprises aplurality of non-contiguous intervals of time during which the secondalternating electric field at the second frequency is applied to thecancer cells, wherein the plurality of non-contiguous intervals of timecollectively add up to at least one week.

Disclosed are methods of altering the electric impedance to analternating electric field in a target site or tumor target site of asubject, comprising applying a first alternating electric field at afirst frequency to the target site or tumor target site for a firstperiod of time, wherein application of the first alternating electricfield at the first frequency to the target site or tumor target site forthe first period of time increases permeability of cell membranes of thecells present in the target site or tumor target site; introducing ananoparticle to the target site or tumor target site, wherein theincreased permeability of the cell membranes enables the nanoparticle tocross the cell membranes; and applying a second alternating electricfield at a second frequency to the target site or tumor target site fora second period of time, wherein the second frequency is different fromthe first frequency, and wherein the impedance in the target site ortumor target site of the subject of the second alternating electricfield at the second frequency is altered, wherein the target site ortumor target site is adjacent to one or more cancer cells, previouslycomprised one or more tumor cells, or is suspected of comprising one ormore tumor cells. In some aspects, the current density and/or power lossdensity in the target site or tumor target site of the subject to thealternating current is altered. In some aspects, the second alternatingelectric field is a tumor-treating field. In some aspects, the cells arecancer cells. In some aspects, the cancer cells are glioblastoma cells,uterine sarcoma cells, breast adenocarcinoma cells, pancreatic cancercells, non-small cell lung cancer, hepatocellular, gastric cancer cells,or brain cancer cells. In some aspects, the cancer cells compriseglioblastoma cells, the first frequency is between 250 kHz and 350 kHz,and the second frequency is between 150 kHz and 250 kHz. In someaspects, the cancer cells comprise uterine sarcoma cells, the firstfrequency is between 125 kHz and 175 kHz, and the second frequency isbetween 75 kHz and 125 kHz. In some aspects, the cancer cells comprisebreast adenocarcinoma cells, the first frequency is between 75 kHz and175 kHz, and the second frequency is between 100 kHz and 300 kHz. Insome aspects, the step of introducing the nanoparticle begins at a giventime, and wherein the step of applying the first alternating electricfield ends at least 12 hours after the given time. In some aspects, thestep of applying the first alternating electric field begins at leastone hour before the given time. In some aspects, the second period oftime comprises a plurality of non-contiguous intervals of time duringwhich the second alternating electric field at the second frequency isapplied to the cancer cells, wherein the plurality of non-contiguousintervals of time collectively add up to at least one week.

Also discussed herein are methods of using heat, or hyperthermia, tokill or ablate cells in a target site or tumor target cite. For example,the methods disclosed herein can use one or more of the nanoparticlesdisclosed herein, wherein the nanoparticles are introduced into a cellin a target site or tumor target site, and then exposed to analternating electric field or alternating magnetic field (AMF). Exposureof the cells in the target site or tumor target site to the alternatingelectric field or magnetic field (AMF) can cause the nanoparticles toheat (e.g. hit temperatures exceeding 100 degrees Fahrenheit), which canresult in the killing the cells in the target site or tumor target site.

Disclosed are methods of killing or ablating cells in a target site ortumor target site with one frequency that allows nanoparticles to enterinto cells in the target site or tumor target site and then applying analternating electric field or alternating magnetic field to the targetsite or tumor target site, wherein the nanoparticles convert thealternating electric field or alternating magnetic field into thermalenergy, thereby killing or ablating the cells in the target site ortumor target site. In some aspects, the methods disclosed herein cafurther comprise applying multiple first and second frequencies.

For example, disclosed are methods of ablating or killing cells in atarget site or tumor target site of a subject, comprising applying afirst alternating electric field at a first frequency to the target siteor tumor target site for a first period of time, wherein application ofthe first alternating electric field at the first frequency to thetarget site or tumor target site for the first period of time increasespermeability of cell membranes of the cells present in the target siteor tumor target site; introducing a nanoparticle to the target site ortumor target site, wherein the increased permeability of the cellmembranes enables the nanoparticle to cross the cell membranes; andapplying a second alternating electric field at a second frequency or analternating magnetic field to the target site or tumor target site for asecond period of time, wherein one or more cells present in the targetsite or tumor target site are killed or ablated. In some aspects, thesecond alternating electric field is a tumor-treating field. In someaspects, the target site or tumor target site is adjacent to one or morecancer cells, previously comprised one or more tumor cells, or issuspected of comprising one or more tumor cells. In some aspects, thecurrent density and/or power loss density in the target site or tumortarget site of the subject to the alternating current is altered. Insome aspects, the second alternating electric field is a tumor-treatingfield.

In any of the methods disclosed herein, the methods can further compriseadministering to the subject an anticancer drug, a cytotoxic drug,pain-management drug, pseudomonas exotoxin A, a non-radioactive isotope(e.g. boron-10 for boron neutron capture therapy), a photosensitizer(e.g. photofrin, foscan, 5-aminolevulinic acid, Mono-L-aspartyl chlorine6, pthalocyanines, Meta-tetra(hydroxyphenyl)porphyrins, texaphyrins,Tin ethyl etipurpurin), or applying or exposing the subject to anelectronic system for influencing cellular functions. For example, inany of the methods disclosed herein, a subject can be exposed to or asystem can be applied to the subject wherein the system includes one ormore controllable low energy HF (High Frequency) carrier signalgenerator circuits, one or more data processors for receiving controlinformation, one or more amplitude modulation control generators and oneor more amplitude modulation frequency control generators. In someaspects, the amplitude modulation frequency control generators areadapted to accurately control the frequency of the amplitude modulationsto within an accuracy of at least 1000 ppm, most preferably to withinabout 1 ppm, relative to one or more determined or predeterminedreference amplitude modulation frequencies. Additional embodiments andspecific frequencies for particular cancers are described in U.S. Pat.No. 8,977,365, which is hereby incorporated by reference in its entiretyfor it teaching of systems and methods useful for influencing cellularfunctions or malfunctions in a subject.

D. Increasing Antitumor Activity of TTF by Altering Electric Field'sDistribution Utilizing Nanoparticles

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased, wherein the magnitude of thecurrent density of the alternating electric field is increased in thetarget site.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased, wherein the impedance in thetarget site is lowered.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased, wherein the conductivity in thetarget site is increased and/or wherein the impedance in the siteadjacent to the target site is increased.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased, wherein the alternatingelectric field is a tumor-treating field.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the efficacy of the alternating electric field in thetarget site of the subject is increased, wherein the target site is atumor target site.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; introducing a conductive nanoparticleto a target site in the subject; applying an alternating electric fieldto the target site and the site adjacent to the target site of thesubject, wherein the increased efficacy of the alternating electricfield in the target site results in an increased anti-mitotic effect ofthe alternating electric field in the target site.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the nanoparticle isa non-conductive nanoparticle.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the impedance inthe non-target site adjacent to the target site is increased and/orwherein the conductivity in the non-target site adjacent to the targetsite is decreased.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the impedance inthe target site is lowered and/or wherein the conductivity in the targetsite is increased.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the magnitude ofthe current density of the alternating electric field is decreased inthe non-target site adjacent to the target site.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, further comprisingintroducing a conductive nanoparticle to the target site in in thesubject. In some aspects, the impedance in the target site is lowered.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the alternatingelectric field is a tumor-treating field.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the efficacy of the alternating electric field inthe target site of the subject is increased, wherein the target site isa tumor target site. In some aspects, the increased efficacy of thealternating electric field in the target site results in an increasedmitotic effect of the alternating electric field in the target site. Insome aspects, the nanoparticle is introduced into the tumor. In someaspects, the nanoparticle is introduced into the tumor via injectionpost primary tumor resection. In some aspects, the nanoparticle isintroduced into the tumor via intratumor injection (e.g. computedtomography-guided, during surgery or biopsy).

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle is introduced intratumorally,intracranially, intraventricularly, intrathecally, epidurally,intradurally, intravascularly, intravenously (targeted or non-targeted),intraarterially, intramuscularly, subcutaneously, intraperitoneally,orally, intranasally, via intratumor injection (e.g. computedtomography-guided, during surgery or biopsy) or via inhalation.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle is introduced to the subject in atargeted or non-targeted manner.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle is introduced at about 0.001 to0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600to 700, 700 to 800, 800 to 900, or 900 to 1000 ng per mm³ tumor.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle is introduced at about 0.001 to0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600to 700, 700 to 800, 800 to 900, or 900 to 1000 μg.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle is introduced once, twice, threeor more times.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the conductive nanoparticle comprises or consistsof carbon gold, ferrous iron, selenium, silver, copper, platinum, ironoxide, graphene, iron dextran, superparamagnetic iron oxide, boron-dopeddetonation nanodiamonds, or a combination thereof. In some aspects, theconductive nanoparticle comprises an alloy selected from Au/Ag, Au/Cu,Au/Ag/Cu, Au/Pt, Au/Fe, Au/Cu or Au/Fe/Cu.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the size of the nanoparticle is between 0.5 nm and100 nm.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the size of the nanoparticle is between 0.5 nm and2.5 nm.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the size of the nanoparticle is greater than 100nm.

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the size of the nanoparticle is between 100 nm and200 nm,

Disclosed herein are methods of increasing the efficacy of analternating electric field in a target site of a subject, the methodcomprising: introducing a non-conductive nanoparticle to a site adjacentto the target site in the subject; applying an alternating electricfield to the site adjacent to the target site or to the target site ofthe subject, wherein the nanoparticle has a three-dimensional shape. Insome aspects, the nanoparticle is a nanocube, nanotube, NanoBipyramid,NanoPlate, NanoCluster, Nanochaine, NanoStar, NanoShuttle, NanoHollow,dendrimer, nanorod, nanoshell, nanocage, nanosphere, nanofiber, ornanowire, or a combination thereof. In some aspects, the nanoparticle ismesoporous or nonporous. In some aspects, the nanoparticle is coatedwith a polysaccharides, poly amino acid, or synthetic polymer.

In some aspects, the nanoparticle is incorporated into a scaffold priorto introducing the nanoparticle to the subject.

In some aspects, the nanoparticles are loaded onto or within a scaffold.

In some aspects, the nanoparticle is provided in a pharmaceuticalcomposition.

In some aspects, the pharmaceutical composition further comprises achemotherapeutic agent.

In some aspects, the nanoparticle is conjugated to one or more ligands.In some aspects, the one or more ligands are be conjugated to thenanoparticle via a linker. In some aspects, the linker comprises a thiolgroup, a C2 to C12 alkyl group, a C2 to C12 glycol group or a peptide.In some aspects, the linker comprises a thiol group represented by thegeneral formula HO—(CH)n,—S—S—(CH2)m-OH wherein n and m areindependently between 1 and 5.

In some aspects, the one or more ligands are a small molecule, nucleicacid, carbohydrate, lipid, peptide, antibody, antibody fragment, or atherapeutic agent. In some aspects, the one or more ligands are ananticancer drug or a cytotoxic drug.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a nanoparticle to a target site in the subject; applying analternating electric field to the target site of the subject, whereinthe efficacy of the alternating electric field in the target site of thesubject is increased.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a tumor target site of a subject, the methodcomprising introducing a nanoparticle to a tumor target site in thesubject; applying an alternating electric field to the tumor target siteof the subject, wherein the efficacy of the alternating electric fieldin the tumor target site of the subject is increased.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a tumor target site of a subject, the methodcomprising introducing a nanoparticle to a tumor target site in thesubject; applying an alternating electric field to the tumor target siteof the subject, wherein the efficacy of the alternating electric fieldin the tumor target site of the subject is increased, wherein the tumortarget site is adjacent to one or more cancer cells, previouslycomprised one or more tumor cells, or is suspected of comprising one ormore tumor cells.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a nanoparticle to a target site in the subject; applying analternating electric field to the target site of the subject, whereinthe efficacy of the alternating electric field in the target site of thesubject is increased, wherein the magnitude of the current density ofthe alternating electric field is increased in the target site.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a tumor target site of a subject, the methodcomprising introducing a nanoparticle to a tumor target site in thesubject; applying an alternating electric field to the tumor target siteof the subject, wherein the efficacy of the alternating electric fieldin the tumor target site of the subject is increased, wherein themagnitude of the current density of the alternating electric field isincreased in the tumor target site.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a tumor target site of a subject, the methodcomprising introducing a nanoparticle to a tumor target site in thesubject; applying an alternating electric field to the tumor target siteof the subject, wherein the efficacy of the alternating electric fieldin the tumor target site of the subject is increased, wherein themagnitude of the current density of the alternating electric field isincreased in the tumor target site, wherein the tumor target site isadjacent to one or more cancer cells, previously comprised one or moretumor cells, or is suspected of comprising one or more tumor cells.

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a nanoparticle to a target site in the subject; applying analternating electric field to the target site of the subject, whereinthe efficacy of the alternating electric field in the target site of thesubject is increased and further comprising introducing a non-conductivenanoparticle to a non-target site adjacent to the target site in thesubject. In some aspects, the target site is a tumor target site. Insome aspects, the tumor target site is adjacent to one or more cancercells, previously comprised one or more tumor cells, or is suspected ofcomprising one or more tumor cells

Disclosed are methods of increasing the efficacy of an alternatingelectric field in a target site of a subject, the method comprisingintroducing a non-conductive nanoparticle to a site adjacent to a targetsite in the subject; applying an alternating electric field to thetarget site of the subject, wherein the efficacy of the alternatingelectric field in the target site. of the subject is increased. In someaspects, the target site is a tumor target site. In some aspects, thetumor target site is adjacent to one or more cancer cells, previouslycomprised one or more tumor cells, or is suspected of comprising one ormore tumor cells.

In some aspects, the nanoparticle is a conductive nanoparticle. Aconductive nanoparticle can increase conductivity and lower impedance inthe target site or tumor target site. Thus, in some aspects of thedisclosed methods, the impedance in the target site or tumor target siteis lowered and/or the conductivity in the target site or tumor targetsite is increased.

In some aspects, the nanoparticle is a non-conductive nanoparticle. Insome aspects, the non-conductive nanoparticle is not a ferroelectricnanoparticle. A non-conductive nanoparticle can decrease conductivityand increase impedance in the target site or tumor target site. Thus, insome aspects of the disclosed methods, the impedance in the target siteor tumor target site is increased and/or the conductivity in the targetsite or tumor target site is decreased. In some aspects, the impedancein a site adjacent to the in the target site or tumor target site isincreased and/or the conductivity in the site adjacent to the targetsite or tumor target site is decreased.

In some aspects, any of the nanoparticles disclosed herein can be usedin the methods of increasing the efficacy of an alternating electricfield in the target site or tumor target site of a subject disclosedherein.

In some aspects, the alternating electric field is a tumor-treatingfield as disclosed herein.

In some aspects, the alternating electric fields can be applied for avariety of different intervals ranging from 0.5 hours to 72 hours. Insome aspects, a different duration can be used (e.g., between 0.5 hoursand 14 days). In some aspects, application of the alternating electricfields can be repeated periodically. For example, the alternatingelectric fields can be applied every day for a two hour duration.

In some aspects, the increased efficacy of the alternating electricfield in the target site or tumor target site results in an increasedmitotic effect of the alternating electric field in the target site ortumor target site. For example, the increased mitotic effect can referto interference with proper micro-tubule assembly during metaphase whichcan eventually destroy the cells (e.g. cancer cells) present in or atthe target site or tumor target site during telophase, cytokinesis, orsubsequent interphase.

In some aspects, the magnitude of the current density of the alternatingelectric field is decreased in a site adjacent to the target site ortumor target site.

E. Increasing Uptake of Nanoparticles

Disclosed are methods for improving the transport of a nanoparticleacross a cell membrane of a cell, the method comprising applying analternating electric field to the cell for a period of time, whereinapplication of the alternating electric field increases permeability ofthe cell membrane; and introducing the nanoparticle to the cell, whereinthe increased permeability of the cell membrane enables the nanoparticleto cross the cell membrane. In some aspects, the cells are cancer ortumor cells. In some aspects, the cells are not cancer or tumor cells.

In some aspects, the methods of PCT/US19/40479 filed on Jul. 3, 2019 canbe used in the methods disclosed herein. For example, PCT/US19/40479filed on Jul. 3, 2019 describes methods and processes to deliver asubstance across a cell membrane of a cell, which is hereby incorporatedby reference for its teaching of same. For example, PCT/US19/40479 filedon Jul. 3, 2019 describes methods and processes to that comprisesapplying an alternating electric field to the cell for a period of time,wherein application of the alternating electric field increasespermeability of the cell membrane; and introducing the substance to avicinity of the cell, wherein the increased permeability of the cellmembrane enables the substance to cross the cell membrane.

In some aspects, the nanoparticle delivered across a cell membrane of acell is a conductive nanoparticle. In some aspects, the nanoparticle isa non-conductive nanoparticle. In some aspects, the non-conductivenanoparticle is not a ferroelectric nanoparticle. Thus, in some aspectsthe nanoparticle can be a non-ferroelectric nanoparticle.

In some aspects, any of the nanoparticles disclosed herein can be usedin the disclosed methods for improving the transport of a nanoparticleacross a cell membrane of a cell.

In some aspects, thee methods for improving the transport of ananoparticle across a cell membrane of a cell, the method comprises, inpart, applying an alternating electric field to the cell for a period oftime. In some aspects, an alternating electric field can be used tointroduce the nanoparticle into cancer cells only. In some aspects, thealternating electric field can be applied at a frequency of about 200kHz. In some aspects, the alternating electric field can be applied at afrequency between 50 and 190 kHz. In some aspects, the alternatingelectric field can be applied at a frequency between 210 and 400 kHz. Insome aspects, the alternating electric field has a field strength of atleast 1 V/cm RMS. In some aspects, the alternating electric field has afrequency between 50 and 190 kHz. In some aspects, the alternatingelectric field has a frequency between 210 and 400 kHz. In some aspects,the alternating electric field has a field strength of at least 1 V/cmRMS. In some aspects, the alternating electric field has a fieldstrength between 1 and 4 V/cm RMS.

In some aspects, the step of introducing the nanoparticle begins at agiven time, and wherein the step of applying the alternating electricfield ends at least 12 hours after the given time. In some aspects, thestep of applying the alternating electric field begins at least one hourbefore the given time. In some aspects, the step of applying thealternating electric field begins at least one to around twenty-fourhours before the given time.

F. Increasing Uptake of Nanoparticles and Treating

Disclosed are methods of increasing cell permeability of a cell (e.g. atumor or cancer cell) with one frequency that allows nanoparticles in tothe cell and then applying a second frequency for treatment via tumortreating fields based on the presence of the nanoparticle in the cell.In some aspects, the methods disclosed herein ca further compriseapplying multiple first and second frequencies. In some aspects, thefirst frequency can selected so as to maximize the openings in the cellmembrane such that nanoparticles can pass through and the. In someaspects, the second frequency can be chosen to enhance the effect of aTTField on the cell.

Disclosed are methods for reducing the viability of a cell, the methodcomprising: applying a first alternating electric field at a firstfrequency to the cell for a first period of time, wherein application ofthe first alternating electric field at the first frequency to the cellfor the first period of time increases permeability of cell membrane ofthe cancer cell; introducing a nanoparticle to the cell, wherein theincreased permeability of the cell membrane of the cell enables thenanoparticle to cross the cell membrane; and applying a secondalternating electric field at a second frequency to the cell for asecond period of time, wherein the second frequency is different fromthe first frequency, and wherein the second alternating electric fieldat the second frequency reduces viability of the cell.

Disclosed are methods for reducing the viability of cancer cells, themethod comprising: applying a first alternating electric field at afirst frequency to the cancer cells for a first period of time, whereinapplication of the first alternating electric field at the firstfrequency to the cancer cells for the first period of time increasespermeability of cell membranes of the cancer cells; introducing ananoparticle to the cancer cells, wherein the increased permeability ofthe cell membranes enables the nanoparticle to cross the cell membranes;and applying a second alternating electric field at a second frequencyto the cancer cells for a second period of time, wherein the secondfrequency is different from the first frequency, and wherein the secondalternating electric field at the second frequency reduces viability ofthe cancer cells.

Disclosed are methods for reducing the viability of a cell, the methodcomprising: applying a first alternating electric field at a firstfrequency to a target site or tumor target site comprising the cell fora first period of time, wherein application of the first alternatingelectric field at the first frequency to the target site or tumor targetsite for the first period of time increases permeability of cellmembranes of the cell; introducing a first nanoparticle to the cell,wherein the increased permeability of the cell membranes enables thefirst nanoparticle to cross the cell membrane; and applying a secondalternating electric field at a second frequency to the target site ortumor target site for a second period of time, wherein the secondfrequency is different from the first frequency, and wherein the secondalternating electric field at the second frequency reduces viability ofthe cell, and further comprising introducing a second nanoparticle to asite adjacent to the target site or tumor target site in the subject. Insome aspects, the site adjacent to the target site or tumor target sitein the subject can be any site adjacent to cell that do not containcell. For example, the site adjacent to the target site or tumor targetsite in the subject can be any site adjacent to a cancer or tumor cellthat do not contain the cancer or tumor cell.

Disclosed are methods for reducing the viability of cancer cells, themethod comprising: applying a first alternating electric field at afirst frequency to the cancer cells for a first period of time, whereinapplication of the first alternating electric field at the firstfrequency to the cancer cells for the first period of time increasespermeability of cell membranes of the cancer cells; introducing a firstnanoparticle to the cancer cells, wherein the increased permeability ofthe cell membranes enables the first nanoparticle to cross the cellmembranes; and applying a second alternating electric field at a secondfrequency to the cancer cells for a second period of time, wherein thesecond frequency is different from the first frequency, and wherein thesecond alternating electric field at the second frequency reducesviability of the cancer cells, and further comprising introducing asecond nanoparticle to a non-target site adjacent to the cancer cells inthe subject. In some aspects, the non-target site can be any siteadjacent to cancer cells that do not contain cancer cells.

In some aspects, the nanoparticle or first nanoparticle is a conductivenanoparticle. A conductive nanoparticle can increase conductivity andlower impedance in the cell, target site or tumor target site. Thus, insome aspects of the disclosed methods, the impedance in the cell, targetsite or tumor target site is lowered and/or the conductivity in thecell, target site or tumor target site is increased.

In some aspects, the second nanoparticle is a non-conductivenanoparticle. In some aspects, the non-conductive nanoparticle is not aferroelectric nanoparticle. A non-conductive nanoparticle can decreaseconductivity and increase impedance in the cell, target site or tumortarget site. Thus, in some aspects of the disclosed methods, theimpedance in the non-target site adjacent to the cell, target site ortumor target site is increased and/or the conductivity in the siteadjacent to the cell, target site or tumor target site is decreased.

In some aspects, the current density and/or power loss density in in thecell, target site or tumor target site to the alternating current can bealtered. In some aspects, the current density in the cell, target siteor tumor target site is increased. In some aspects, the current densityis in the cell, target site or tumor target site decreased. In someaspects, power loss density in the cell, target site or tumor targetsite is increased. In some aspects, power loss density in the cell,target site or tumor target site is decreased.

In some aspects, the second period of time comprises a plurality ofnon-contiguous intervals of time during which the second alternatingelectric field at the second frequency is applied to the cancer cells,wherein the plurality of non-contiguous intervals of time collectivelyadd up to at least one week.

In some aspects, the cells are disposed in a body of a living subject,wherein the first alternating electric field is applied to the cells(e.g. tumor or cancer cells) by applying a first alternating electricfield to the subject's body, the second alternating electric field isapplied to the cells by applying a second alternating electric field tothe subject's body, and wherein the introducing comprises administeringthe nanoparticle to the subject. In some aspects, the application of thefirst and second alternating fields occurs at a location on thesubject's body based on the type and location of a cancer in thesubject. For example, for glioblastoma the first and second alternatingfields can be applied to the head.

In some aspects, the first alternating electric field has a fieldstrength of at least 1 V/cm RMS. In some aspects, the first alternatingelectric field has a field strength between 1 and 4 V/cm RMS.

In some aspects, the disclosed methods further comprise introducing asecond nanoparticle to a site adjacent to the cancer cells in thesubject. In some aspects, the site can be any site adjacent to thecancer cells that do not contain cancer cells. In some aspects, thesecond nanoparticle is a non-conductive nanoparticle. In some aspects,the non-conductive nanoparticle is not a ferroelectric nanoparticle. Anon-conductive nanoparticle can decrease conductivity and increaseimpedance in or at the site adjacent to the cancer cells. Thus, in someaspects of the disclosed methods, the impedance in the site adjacent tothe cancer cell is increased and/or the conductivity in the siteadjacent to the cancer cell is decreased.

G. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for imagingand/or treating. In some aspects, the kit can comprise one or more ofthe disclosed nanoparticles. The kits also can contain equipment forapplying alternating electrical fields.

Disclosed herein are kits comprising one or more of the nanoparticlesdescribed herein in and a device capable of administering an alternatingelectric field. For example, disclosed herein are kits comprising one ormore of the nanoparticles described herein in and a TTFields device (e.gOptune®, Novocure Ltd.).

EXAMPLES Using Nanoparticles to Increase Tumor Connectivity and EnhanceAlternating Electric Fields Intensity in the Tumor

Preclinical studies showed a correlation between Tumor treating fields(TTFields) efficacy in killing cancer cells and field intensity [1]. Arecent study [2] showed that TTFields intensity in tumor regioncorrespond with outcome in newly diagnosed glioblastoma patients. Inthat study, TTFields intensity was calculated utilizing computationalsimulations of patient-specific head models of 317 patients treated withTTFields (at 200 kHz). The dielectric properties assigned to the modelsin these simulations were based on values from the literature [3-4] andthe metric for TTFields intensity was defined as the minimal powerdensity out of the two values originated from each pair of transducerarrays used to deliver TTFields to the patient head (named LMiPD).

In order to demonstrate the influence of enhanced tumor conductivity onTTFields intensity in the tumor, 45 models of patients were utilizedfrom the computational study [2] and the electrical conductivity of thegross tumor volume was increased by 25% (0.3 S/m instead of 0.24 S/m)and the simulations were re-run. The results of these studiesdemonstrate that for all 45 patients, increasing tumor conductivityenhance the average LMiPD in the gross tumor volume by a similar orhigher percentage than the relative increase in conductivity, as shownin FIG. 1A (26%-46%, median=32%, std=4%). Intensity increase wasobserved for various tumor volumes (206-85091 mm³). These resultsindicate that increasing tumor conductivity can result in enhancement ofTTFields efficacy.

A study investigating the influence of gold nanoparticles (GNP) ontissue conductivity has shown that integrating GNP enhanced tissueconductivity [6]. This study reported that at frequency of 10 kHz, theaverage conductivity of minced fat tissue increased from 0.0191 S/m to0.0198 S/ and from 0.55 S/m to 0.57 S/m in minced muscle tissue.

A study investigating the effect of nano-Titanium dioxide (nano-TiO2) onthe signal of Electrical Impedance Tomography (EIT) showed thatinjecting nano-TiO2 to tumors of mice inoculated with tumors at theirarmpits enhanced the EIT signal [7]. This study reports that at 40 kHzthe tumor impedance decreased after the injections of the nano-TiO2particles from 12.5 kOhm to 11.2 kOhm, an increase of 12% inconductivity.

Studies investigating the conductivity of nanoparticles in solutionswere also performed. A study investigating the electrical conductivityof iron oxide nanoparticle dispersed in ethylene glycol-based fluidshowed that the electrical conductivity of the nano-fluid increased from0.39 μS/cm to 2.419 mS/cm for a loading of 4 vol % iron oxide at 25° C.[8].

Polyethylene glycol (PEG) frequently used as encapsulation agent becauseof non-toxic properties, and it can increase the dispersibility ofnanoparticles. PEG is advantageous to prevent agglomeration and toincrease penetration of the nanoparticles into the cells environment. Astudy investigating the dielectric properties of Mn0.5Zn0.5Fe2O4nanoparticles (MNPs) encapsulated by PEG report that MNPs remainconducting after encapsulating at frequency range 5 kHz to 120 kHz. [9]

REFERENCES

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[2] M Ballo, Z Bomzon, N Urman, G Lavy-Shahaf, S A Toms; P01.113Increasing TTFields dose to the tumor bed improves overall survival innewly diagnosed glioblastoma patients, Neuro-Oncology, Volume 20, Issuesuppl_3, 19 Sep. 2018, Pages iii257,https://doi.org/10.1093neuonc/noy139.155

[3] N Urman, S Levy, A Frenkel, A Naveh, H S Hershkovich, E Kirson, CWenger, G Lavy-Shahaf, D Manzur, O Yesharim, Z Bomzon; P04.57 Creatingpatient-specific computational head models for the study oftissue-electric field interactions using deformable templates,Neuro-Oncology, Volume 20, Issue suppl_3, 19 Sep. 2018, Pages iii292,https://doi.org/10.1093/neuonc/noy139.291

[4] Wenger C, Salvador R, Basser P J, Miranda P C. The electric fielddistribution in the brain during TTFields therapy and its dependence ontissue dielectric properties and anatomy: a computational study. PhysMed Biol. 2015; 60(18):7339-57.

[5] Hershkovich H S, Bomzon Z, Wenger C, Urman N, Chaudhry A,Garcia-Carracedo D, Kirson E D, Weinberg U, Wassermann Y, Palti Y.,“First steps to creating a platform for high throughput simulation ofTTFields”, Conf Proc IEEE Eng Med Biol Soc. 2016 August; 2016:2357-2360. doi: 10.1109/EMBC.2016.7591203.

[6] Ostovari M, Riahi Alam, Zabihzadeh, Gharibvand Hoseini-Ghahfarokhi;“The Effect of Gold Nanoparticles on Electrical Impedance of Tissue onLow Frequency Ranges”, J Biomed Phys Eng. 2018 Sep. 1; 8(3):241-250.eCollection 2018 September.

[7] Liu R1, Jin C, Song F, Liu J. “Nanoparticle-enhanced electricalimpedance detection and its potential significance in image tomography”,Int J Nanomedicine. 2013; 8:33-8. doi: 10.2147/IJN.S37275. Epub 2013Jan. 3.

[8] Jamilpanah, P., Pahlavanzadeh, H. & Kheradmand, A. “Thermalconductivity, viscosity, and electrical conductivity of iron oxide witha cloud fractal structure” , Heat Mass Transfer (2017) 53: 1343.https://doi.org/10.1007/s00231-016-1891-5

[9] L. Armitasari et al. “Effect of Polyethylene Glycol (PEG-4000) onDielectric Properties of Mn0.5Zn0.5Fe2O4 Nanoparticles” , 2018 IOP Conf.Ser.: Mater. Sci. Eng. 367 012035.

Barian Titanate Nanoparticles Sensitize Treatment-Resistant BreastCancer Cells to the Antitumor Action of Tumor-Treating Fields

Introduction: Many preclinical and clinical studies indicate thatTTFields would be applicable for other tumor types including breast,lung, pancreatic, and ovarian cancers. Early clinical trials have shownthat only TTFields treatment for GBM patients was not significantlybetter than conventional chemotherapy. However, recent preclinicalstudies suggest that combination therapy of TTFields with conventionaltreatments including chemotherapy, immunotherapy, and radiotherapy aremore effective than TTFields monotherapy in GBM. Despite the promiseshown by TTFields as a viable cancer therapy, not much is known aboutTTFields responsive sensitiser.

Ferroelectric nanomaterials (non-conductive nanoparticles) have emergedas promising tools for enhancing electric stimulation of cells andtissues. Among ferroelectric materials, barium titanate nanoparticles(BTNPs) have high dielectric constants and suitable piezoelectriccharacteristics with high biocompatibility. Recent reports suggest thatBTNPs could be used in a wide range of applications in nanomedicine,including non-linear imaging purposes, drug delivery, tissueengineering, and bio-stimulation. For instance, BTNPs promote higherinternalisation of doxorubicin in human neuroblastoma cells and BTNPswith polyethylenimine have been shown to improve cellular uptake forcell imaging and DNA delivery. Disclosed herein are studies thatinvestigated whether BTNPs could enhance the antitumor action ofTTFields in response to TTFields. The data herein shows that BTNPs aloneare cytocompatible with breast cancer cells, but in response toTTFields, it can sensitize TTField-resistant breast cancer cells to theantitumor action of TTFields. Further, the data herein demonstrates thatBTNPs were taken up by TTFields stimulation and these promoted antitumoraction of TTFields by enhancing cell cycle-related apoptosis in breastcancer cells. Therefore, this study shows a TTField-responsivesensitiser, BTNPs, in breast cancer cells. The studies described hereincan be performed on other cancer cells to show additional evidence thatBTNPs may be taken up by TTFields stimulation and these promotedantitumor action of TTFields by enhancing cell cycle-related apoptosisin other cancer cell types.

Results Characteristics and Cytocompatibility of BTNPs in Breast CancerCells

Since dielectric permittivity of BTNPs can be maximized depending on itssize, two different sizes of FBS (fetal bovine serum) coated BTNPs wereprepared (100 nm and 200 nm). The SEM images of 100 nm and 200 nm BTNPsshowed typical round shape and homogeneous size of the nanoparticles(FIGS. 3A and 3B). The hydrodynamic radius of 100 nm and 200 nm BTNPswere 110±35 nm and 224±63 nm and the Zeta potentials of 100 nm and 200nm BTNPs were −14.1±10.4 mV and −14.5±12.8 mV, respectively (FIG. 3C),indicating that BTNPs were relatively stable in aqueous dispersions.Next, the cytocompatibility of 100 nm and 200 nm BTNPs were examined bycell viability and clonogenic assay in the two breast cancer cell lines,MCF-7 and BT-549. Ethanol was used as a positive control in theseassays. The cell viability assay indicated that treatment with 100 nmand 200 nm BTNPs up to a concentration of 20 μg/ml did not affect cellviability in MCF-7 and BT-549 cells (FIG. 4A and FIG. 4B). In addition,the clonogenic assay showed that treatment with 100 nm and 200 nm BTNPsup to a concentration of 100 μg/ml did not affect colony formation inMCF-7 and BT-549 cells (FIGS. 4C-F). Taken together, these resultsindicate that BTNPs exhibit cytocompatibility with non-cytotoxic effectsin breast cancer cells.

BTNPs Sensitise TTField-Resistant Breast Cancer Cells in Response toTTFields

TTFields efficacy was tested in three breast cancer cell lines, MCF-7,MDA-MB-231, and BT-549. Among these, MCF-7 cells were the most resistantto TTFields (FIG. 5A), which is consistent with previous reports. Thus,the combinatorial effect of BTNPs and TTFields was examined in MCF-7cells. Cell viability and clonogenic assays showed that treatment with100 nm and 200 nm BTNPs enhanced the antitumor action of TTFields inTTField-resistant MCF-7 cells (FIGS. 5B-D). Notably, 200 nm BTNPs weremore potent than the 100 nm ones (FIGS. 5B-D), indicating that size canbe an important factor in the antitumor activity of BTNPs in presence ofTTFields. Thus, these results indicated that BTNPs sensitizeTTField-resistant breast cancer cells in response to TTFields.

TTFields Induce the Cytosolic Accumulation of BTNPs in Breast CancerCells

To investigate the mechanism of this sensitization mediated by BTNPs inpresence of TTFields, whether BTNPs accumulate into breast cancer cellsin response to TTFields was examined. First, a fluorescence-activatedcell sorting (FACS) analysis was performed to determine cell size andgranularity in TTField-treated and BTNP/TTField-treated cells. Theseparameters were similar between the control and TTField-treated MCF-7and BT-549 cells (FIG. 6A and FIG. 6B, left panels). Cell size andgranularity increased in BTNP/TTField-treated MCF-7 and BT-549 cells(FIG. 6A and FIG. 6B, middle and right panels). In addition, methyleneblue staining showed the cytosolic accumulation of BTNPs in response toTTFields in MCF-7 and BT-549 cells (FIG. 6C and FIG. 6D). In addition,transmission electron microscopy (TEM) analysis showed that BTNPsaccumulated in the cytoplasm of in TTField-treated MCF cells (FIG. 6E).Therefore, these results indicated that BTNPs accumulated into thecytoplasm of breast cancer cells in response to TTFields.

TTFields Combined with BTNPs Modulates Cell Cycle-Apoptosis Pathways

To further investigate the regulatory action of the TTFields/BTNPscombination approach, a NanoString nCounter™ Pan-Cancer pathway analysiscontaining probes targeting 700 transcripts related to 13 types ofcancer pathways was carried out in MCF-7 cells exposed to TTFields andtreated without or with 200 nm BTNPs for 48 hrs. MCF-7 cells treatedwith BTNPs with no exposure to TTFields was also included as a control.The gene expression patterns were similar between control andBTNPs-treated MCF-7 cells, while TTFields treatment induced dramaticchanges in 9 different types of cancer pathways (FIG. 7A). Among them,cell cycle-apoptosis, Wnt, transcriptional migration, transforminggrowth factor beta (TGF-β), driver gene, Notch, Janus kinase-signaltransducer and activator of transcription (JAK-STAT), and Ras signallingwere significantly modulated in TTField-treated and BTNP/TTField-treatedMCF-7 cells (FIG. 7B), evidencing that BTNPs/TTFields can have acapacity to modulate several cancer signaling pathways. Cell cyclepathways were further analyzed. The data showed that several cell cycleregulatory transcripts including cyclin dependent kinase 4 (CDK4), RB1,tumor protein TP53, cyclin dependent kinase 6 (CDK6), MDM2, andCDKN1A/2A were modulated in BTNP/TTField-treated MCF-7 cells (FIG. 8A).In addition, Western blot analysis for the cell cycle regulatory genesalso showed that TTFields combined with BTNPs inhibited cell cycleprogression, as determined by significant decreases in CDK6 andtranscription factor E2F1 (FIG. 8B). These results indicate thatTTFields combined with BTNPs exerts anticancer activity on breast cancercells by modulating cancer-related pathways, and specifically inhibitingcell cycle progression.

Discussion

BTNPs had non-cytotoxic effects in breast cancer cells and enhanced theantitumor activity of TTField-resistant breast cancer cells in responseto TTFields. Further, it was found that TTFields triggered theaccumulation of BTNPs, which promoted the cell cycle-related apoptosispathway. These data show that biocompatible nanomaterials such as BTNPscan be used as a TTField-responsive sensitizer in cancer cells.

These results showed that BTNPs had non-cytotoxic effects even at highconcentrations (100 μg/ml) in breast cancer cells, indicating that theseare biocompatible. Consistent with these results, other reports haveshown that treatment with BTNPs have minimal adverse effects, as evidentfrom several assays including metabolic activity,viability/cytotoxicity, early apoptosis, and reactive oxygen species(ROS) generation in multiple types of cells such as human neuroblastomaSH-SY5Y cells, HeLa cells, and rat mesenchymal stem cells. The resultsalso show that treatment with only BTNPs did not significantly alter the13 types of major cancer pathways and cell cycle regulatory proteins(FIG. 7). BTNPs and its coated composites could be used as abiocompatible sensitizer for TTFields.

The data shows that TTFields efficacy is dependent on cell doubling timein various cancer cell lines. However, MCF-7 cells were more resistantto TTFields than MDA-MB-231 and BT-549 cells, despite the similar celldoubling time between MCF-7 and MDA-MB-231 cells. The data shows thatBTNPs sensitized the TTField-resistant MCF-7 breast cancer cells inresponse to TTFields, indicating that the TTField-responsive sensitizerssuch as BTNPs could enhance the efficacy of TTFields inTTField-resistant cancer cells. In this context, several studies showedthat chemotherapeutic agents or radiotherapy enhance the efficacy ofTTFields in various cancer cells. However, these combination treatmentsdo not respond to TTFields, indicating that conventionalchemotherapeutic agents or radiotherapy is not a specific sensitizer forTTFields, which is a physical treatment modality. Therefore, the dataindicates that BTNPs can be used as a TTField-responsive sensitiser forTTField-resistant tumors.

The data shows that BTNPs were accumulated into the cytoplasm of breastcancer cells in response to TTFields. Nanoparticles (NPs)internalization into cells is known to be dependent on particle size andits zeta potential. NPs under 200 nm can be engulfed by cancer cellsthrough clathrin-dependent pathway or macro-pinocytosis pathway.However, specific inhibitors for these pathways, such as chlorpromazine,amiloride, and cytochalasin D, did not modulate the accumulation ofBTNPs in cytoplasm in response to TTFields, indicating that BTNPaccumulation in cytoplasm is not mediated by clathrin-dependent pathwayor macro-pinocytosis pathway. The data herein evidences that increasedmembrane permeability by TTFields can induce BTNP accumulation incytoplasm of cancer cells.

The data herein indicates that TTFields combined with BTNPssignificantly modulated the cell cycle-apoptosis pathways over otherrelated pathways. Since cells with mitotic defects undergo mitoticcatastrophe or G1-arrest senescence, the data shows that TTFieldscombined with BTNPs could induce mitotic catastrophe and G1-arrestsenescence by modulating cell cycle-apoptosis pathway, as evident by thedecrease in G1 cell cycle regulators including CDK4/6, p-RB, and E2F1 inthe BTNPs/TTField-treated cells. In addition to cell cycle-apoptosispathway, significant modulation of several cancer pathways includingWnt, transcriptional migration, transforming growth factor beta (TGF-β),driver gene, Notch, Janus kinase-signal transducer and activator oftranscription (JAK-STAT), and Ras signalling were observed inTTField-treated and BTNPs/TTField-treated MCF-7 cells. The data alsoshows that BTNPs, characterized by their high biocompatibility andferroelectric properties, acts as a TTField-responsive sensitizer tobreast cancer cells by modulating cell cycle-apoptosis pathway (FIG. 9).Therefore, electric field responsive nanomaterials, such as BTNPs, canbe used as a TTField-responsive sensitizer to enhance the therapeuticefficacy of TTFields in cancer cells.

Methods

Cell culture: MCF-7 and BT-549 breast cancer cell lines were purchasedfrom American Type Culture Collection (ATCC, Manassas, Va.). Asconfirmed by the information provided by ATCC, both cell lines wereauthenticated by their karyotypes, images, and detailed gene expression.Both cell lines were preserved and passaged in less than 2 months inaccordance with ATCC protocols, and tested for mycoplasma infection bypolymerase chain reaction (PCR) once a week. MCF-7 cells were culturedin Dulbecco's Modified Eagle Media (DMEM, Corning, N.Y., USA) and BT-549cells were cultured in RPMI (Corning, N.Y., USA). All media types weresupplemented with 10% fetal bovine serum (FBS, Corning, N.Y., USA) and1% penicillin/streptomycin (Sigma-Aldrich, MO, USA). All cell lines weremaintained in a humidified 5% CO2 incubator at 37° C.

TTFields application: MCF-7 (1.5×10⁴) and BT-549 (1×10⁴) cells wereseeded on 18 mm glass coverslips (Marienfeld-Superior, Mediline,Lauda-Königshofen, Germany) or 22 mm plastic coverslips (Thermo FisherScientific, MA, USA) for 24 hrs and those coverslips were transferred toceramic inovitro dishes (NovoCure, Haifa, Israel) using autoclavedforceps. For TTFields treatment, we applied the inovitro system(NovoCure, Haifa, Israel) for 72 hrs as described previously. Briefly,cells on a coverslip were exposed to 1 V/cm at 150 kHz with a current of150 mA generated by inovitro TTFields generators (NovoCure, Haifa,Israel) and the plate temperature was maintained at 37° C. by arefrigerated incubator (ESCO Technologies, USA) at 19° C.

Generation and physicochemical characterization of BTNPs: Bariumtitanate nanoparticles (100 nm, 200 nm) were purchased from US ResearchNanomaterials Inc. (TX, USA) and used without further purification.BTNPs were dispersed in ethanol and sonicated to mitigate aggregation.In addition, 5% FBS was added to coat the surface of BTNPs with aprotein corona, before addition to cells. The nanostructures andmorphologies of prepared BTNPs were examined by field emission scanningelectron microscopy (FE-SEM) with a Sirion-400 (FEI, OR, USA) and TEMwith a JEM-2100F (JEOL, Japan). The zeta-potential of FBS coated BTNPswere measured by dynamic light scattering in a Zetasizer Nano ZS(Malvern Instruments Ltd., UK).

Cell viability assay: Cell viability assays were performed using WST-8reagent (Cyto X; LPS solution, Daejeon). MCF-7 cells (0.5×10⁴) wereseeded on a 96-well plate and treated with media containing increasingconcentrations of BTNPs or ethanol as a vehicle control. After 72 hrs,WST-8 reagent (10 μl) was added to each well and the plate incubated for2 hrs at 37° C. Subsequently, the absorbance was measured at 450 nmusing a VersaMax Microplate Reader (Molecular Devices, CA, USA).

Clonogenic assay: The clonogenic assay was performed as describedpreviously. MCF-7 or BT-549 cells (500 in number) were seeded on a 22 mmplastic coverslip in a 6-well plate for 24 hrs. Using autoclavedforceps, the coverslips were transferred to ceramic inovitro dishes andincubated with inovitro TTFields generators for 72 hrs. After TTFieldstreatment, the coverslips were transferred to a 6-well plate andincubated at 37° C. After 7 days, colonies were fixed and stained with1% crystal violet (Sigma-Aldrich) and 40% methanol solution, and thenumber of colonies counted.

Cell counting: To evaluate the number of alive cells in the same volume,absolute cell counts were acquired using a BD Accuri™ C6 flow cytometer(BD Biosciences, CA, USA) as described previously. Briefly, detachedMCF-7 and BT-549 cells in fresh media (500 μl) were stained withpropidium iodide (50 μg/ml; PI; Sigma-Aldrich, MO, USA) and the numberof cells in PI-negative population was counted in a 100 μl volume.

Cell cycle analysis: Cell cycle analysis was performed. Briefly, thecells treated with conditions described above were trypsinised, washedtwice in PBS, and fixed with ice-cold 70% ethanol. Fixed cells wereincubated with PI (50 μg/ml) and RNase (100 μg/ml) at 37° C. for 30 minand then analyzed with a BD Accuri™ C6 flow cytometer (BD Biosciences,CA, USA).

Apoptosis analysis: Detached cells were collected and apoptosis detectedusing the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, CA,USA) following the manufacturer's protocol. The samples were analyzedusing a BD Accuri™ C6 flow cytometer (BD Biosciences, CA, USA).

Methylene blue staining: Cells were fixed with 4% paraformaldehyde, andthen stained with 0.1% methylene blue (Sigma-Aldrich, MO, USA) dissolvedin Dulbecco's phosphate-buffered saline (DPBS) for 5 min. After washingseveral times with DPBS, the slides were mounted in glycerol and imagesobtained using an LSM 710 confocal microscope (Carl Zeiss Inc.,Germany).

TEM imaging: TTFields treated MCF-7 cells with and without BTNPtreatment were detached and fixed in 2.5% glutaraldehyde (Sigma-Aldrich,MO, USA) and 0.1 M phosphate buffer (pH 7.3) at 4° C. overnight. Afterthis fixation, the cells were treated with 1% osmium tetroxide and 1.5%potassium ferrocyanide in 0.1 M phosphate buffer (pH 7.3) for 1 h at 4°C. in dark. Subsequently, these were embedded in Epon 812(Sigma-Aldrich, MO, USA) after dehydration in a treatment cycle ofethanol and propylene oxide. The polymer reaction was carried out byusing pure resin at 70° C. for two days. Ultrathin samples were obtainedwith an UltraCut-UCT ultramicrotome (Leica, Austria) and collected on150 mesh copper grids. After staining with 2% uranyl acetate for 10 minand lead citrate for 5 min, the samples were examined at 120 kV in aTecnai G2 Spirit Twin TEM setup.

RNA isolation and NanoString analysis: Total RNA was isolated usingQIAzol reagents (Qiagen, Hilden, Germany) from treated cells. Followingthe procedures provided by the nCounter XT CodeSet Gene ExpressionAssays (NanoString Technologies, WA, USA), 100 ng of RNA was used tohybridize with probes.

Western blot analysis: Western blotting was performed as describedpreviously. Briefly, proteins were separated by SDS-polyacrylamide gelelectrophoresis, transferred to a nitrocellulose membrane, and detectedusing specific antibodies. The following antibodies were used: rabbitpolyclonal anti-CDK4, rabbit monoclonal CDK6, rabbit monoclonalphospho-RB, rabbit polyclonal RB (Santa Cruz Biotechnology, CA, USA);mouse monoclonal p21, mouse monoclonal E2F1, mouse monoclonal MDM2,mouse polyclonal anti-β-actin (Santa Cruz Biotechnology, CA, USA), andmouse monoclonal p53 (Merck, NJ, USA). Blots were developed usingperoxide-conjugated secondary antibody and visualized with an enhancedchemiluminescence detection system (Amersham Life Science,Buckinghamshire, UK).

Statistical analysis: The two-tailed Student's t-test was performed toanalyze statistical differences between groups. P-values of less than0.05 were considered statistically significant. Statistical analyseswere performed using Microsoft Excel and XLSTAT software.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method of altering the electric impedance to an alternatingelectric field in a target site of a subject, comprising a. introducinga conductive nanoparticle to a target site in the subject; and b.applying an alternating electric field to the target site of thesubject, wherein the electric impedance in the target site of thesubject to the alternating current is altered.
 2. The method of claim 1,wherein the current density and/or power loss density in the target siteof the subject to the alternating current is altered.
 3. The method ofclaim 1, wherein the impedance in the target site is lowered.
 4. Themethod of claim 1, wherein the conductivity in the target site isincreased.
 5. The method of claim 1, further comprising: introducing anon-conductive nanoparticle to a site adjacent to the target site in thesubject; and applying an alternating electric field to the site adjacentto the target site of the subject,
 6. The method of claim 4, wherein thecurrent density and/or power loss density in the target site of thesubject to the alternating current is altered.
 7. The method of claim 4,wherein the conductivity is decreased in the site adjacent to the targetsite.
 8. The method of claim 4, wherein the impedance in the siteadjacent to the target site is increased.
 9. The method of claim 4,wherein the conductivity is increased in the target site.
 10. The methodof claim 4, wherein the impedance in the target site is decreased. 11.The method of claim 4, wherein the non-conductive nanoparticle is not aferroelectric nanoparticle.
 12. The method of claim 4, wherein theimpedance in the target site is increased.
 13. The method of claim 4,wherein the conductivity in the target site is decreased.
 14. The methodof claim 4, wherein the alternating electric field is a tumor-treatingfield.
 15. The method of claim 4, wherein the nanoparticles arenanoparticles that increase tissue permittivity.
 16. The method of claim4, wherein the target site is a tumor target site.
 17. The method ofclaim 4, wherein the altered electric impedance in the tumor target siteof the subject to the alternating current results in an increasedmitotic effect of the alternating electric field in the tumor targetsite.